Interleukin-2 (IL-2) is the prototypic T-cell growth factor but elimination of IL-2 results in severe immune dysregulation and systemic autoimmunity because the obligatory function of this cytokine is to maintain CD4+ CD25+ FoxP3+ regulatory T-lymphocytes (Treg). Defining the role of IL-2 in the maintenance of Treg, and how effector and memory cells are generated in the absence of IL-2, is fundamental for understanding the biology of this cytokine and the pathogenesis of diseases caused by its absence (and more generally, by the absence of Treg), and also for developing optimal strategies for using this cytokine and its antagonists as therapeutic immune response modifiers. We will address these issues along the following specific aims. 1. What mechanisms underlie the obligatory role of IL-2 in the maintenance of functional Treg? To test the hypothesis that the obligatory functions of IL-2 are to prevent apoptotic death of Treg and to promote expression of genes involved in Treg function, we will define the phenotypic and functional characterisitics of Treg that develop in the periphery and the thymus in the absence of IL-2 signals. We will use the genetic approach of deleting Bim, the sensor of cytokine deprivation, to generate viable Treg in the absence of IL-2. We will define the functions of these cells in vitro and in vivo, and analyze the biochemical pathways that are defective in these cells compared to FoxP3+ cells that develop in the presence of IL-2. Using retroviral gene transduction, we will examine the biochemical signals that link IL-2 to Treg survival and function, particularly Akt and Stat5. We will ask if different amounts and duration of IL-2 signal differentially affect effector/memory cells vs Treg, in order to optimize strategies for therapeutic use of IL-2 and its antagonists. 2. What is the role of IL-2 in effector and memory responses, and how do these responses develop in the absence of IL-2? To test the hypothesis that IL-2 functions to provide activated T-cells the capacity to overcome Treg control, and responses become IL-2-independent in the absence of Treg, we will compare the activation of normal and CD25-deficient T-cells in normal recipients and in IL-2/CD25xCD28 double-knockout Treg-deficient mice we have developed that have a full lymphoid system. We will also define the biochemical correlates of T-cell activation in the presence and absence of Treg and IL-2. We will examine the early events after T-cell recognition of antigen in the absence of Treg, using in vivo imaging methods. 3. What is the basis of the immunological disease associated with IL-2 deficiency? The autoimmune disease of IL-2-knockout BALB/c mice is characterized by rapid-onset hemolytic anemia and lymphoproliferation, and is associated with markedly enhanced IFN-g production (Th1 responses) and activation of dendritic cells (DCs). We will define the mechanism of the enhanced T-cell cytokine response and its contribution to the autoimmune disease, and explore the possibility that DCs are spontaneously activated b5cause of the absence of endogenous Treg. Thus, this project addresses fundamental issues of the functions of IL-2 and how its dual role in activating effector/memory responses and Treg is orchestrated. These studies will shed light on the mechanisms of immune regulation and tolerance, and may help to optimize protocols for the therapeutic uses of IL-2 and its antagonists.7. PROJECT NARRATIVE: Interleukin-2 (IL-2) is a T-cell growth factor that has been used therapeutically to boost immune responses, and IL-2 antagonists have been used as immunosuppressive agents to prevent rejection of allografts and treat graft-vs-host disease. Recent results have shown that IL-2 has a dual biological role it stimulates immune responses by promoting the development of effector and memory cells, and it limits the same responses by virtue of its essential role in the maintenance of functional regulatory T cells (Treg). If we are to use IL-2 or its antagonists as therapeutic agents, and understand its role in immune regulation, we must elucidate how its dual functions are orchestrated. This project uses transgenic and gene knockout mouse models to define why IL-2 is required for Treg survival and function, and how effector and memory responses can be induced in its absence. We will also analyze the mechanisms of the autoimmune disease caused by the absence of IL-2, and determine if different amounts and duration of IL-2 administration can be used to differentially activate effector cells and Treg.